High-throughput sequencing (HTS) is a powerful tool for the analysis of RNA molecules (Hafner, et al. Methods, 44:3-12 (2008); Berninger, et al. Methods, 44:13-21 (2008)). HTS allows the detection of single base differences between molecules, the discovery of undefined molecules and the determination of the differences in RNA composition or expression between different samples. For RNA analysis in HTS, sequencing libraries are typically constructed through a multistep process starting with the ligation of adapters to the ends of the RNA followed by reverse transcription (RT) into cDNA and amplified by PCR prior to HTS sequencing. However, bias has been detected in the ligation of adapters to the RNA that results in some members of an RNA library being misrepresented. This has implications for understanding the biological role of members of the library. For example, HTS experiments have misrepresented the quantity of some small RNAs in a mixture from 2 to 50 fold (Hafner, et al. RNA, 17:1697-1712 (2011) and Alon, et al. Genome Research, 21:1506-1511 (2011)). This type of bias can lead to failure to detect or accurately quantitate species of RNAs that has adverse consequences in understanding the biology of a cell or tissue.